The long-term goals of this proposal are to determine the mechanisms of G protein-coupled receptor (GPCR) activation. GPCRs are characterized structurally by the presence of seven transmembrane domains and functionally by the ability to activate a heterotrimeric guanine-nucleotide binding protein (G protein). Receptors in this family transduce the signals for many medically important hormones and neurotransmitters. GPCRs have also been implicated in the pathogenesis of a broad range of diseases including inflammatory, cardiovascular, and infectious diseases. However, their mechanism of activation is not well understood due to the technical difficulties in studying membrane bound receptors in complex multicellular organisms. Therefore, our work has taken advantage of the genetic accessibility of the yeast S. cerevisiae to study the G protein-coupled alpha-factor pheromone receptor. Preliminary studies based on the analysis of dominant-negative and constitutively-active receptor mutants identified the ends of the transmembrane segments as playing a key role in receptor function. The extracellular ends function in ligand binding and the intracellular ends function in G protein activation. Therefore, the specific aims of this proposal will be to define the roles of the ends of the transmembrane domains in receptor function. The experimental procedures will employ mutational analysis to identify the functionally important residues at the ends of the transmembrane segments, and biochemical approaches to map the residues near the binding sites of the ligand and G protein on the receptor. Biochemical crosslinking experiments and genetic suppressor analysis will then be used in a targeted fashion to establish specific points of contact in the receptor that form the binding pockets for the ligand and G protein. Data from this complementary mix of genetics and biochemistry will be integrated to form a molecular model for the mechanisms of receptor activation. The results of these studies will contribute to greater understanding of GPCR signaling and are expected to have important application to the development of new therapeutic approaches for a wide range of human diseases.